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. 2016 Nov 8;291(52):26922–26933. doi: 10.1074/jbc.M116.752063

FIGURE 4.

FIGURE 4.

Regulation of proteasomal turnover is conserved between EWS-FLI1 and FLI1. A, EWS-FLI1 displays fastest turnover. For GPS analysis of EWS-FLI1, EWSR1, and FLI1, reporter constructs and standards were transduced into HEK293T cells and analyzed after 72 h by FACS. B, full-length proteins EWSR1 and FLI1 were additionally treated with 20 μm MG-132 or 20 μg/ml CHX for 8 h. C, immunoprecipitation of EWS-FLI1, EWSR1, and FLI1 proteins. 3xFLAG-tagged versions were co-expressed with HA-ubiquitin for 48 h and stabilized for an additional 5 h with 20 μm MG-132. After immunoprecipitation of tag proteins, the ubiquitin pattern was analyzed by anti-HA antibody. D, mutation of conserved residue K334R stabilizes FLI1 in GPS analysis. HEK293T cells were transduced for 72 h with reporter constructs of FLI1; mutants K172R, K252R, and K334R; and standards. The EGFP/DsRed ratio was analyzed by FACS. E, turnover-dependent lysine residue is conserved within most ETS family members. Shown is a ClustalW alignment of the conserved ETS domain sequence; the Lys-334/Lys-380 residue is marked in red. IP, immunoprecipitation; IB, immunoblotting; EF, EWS-FLI1; d1h, d4h, and d24h, degron (d) motifs with half-lives of 1, 4, and 24 h, respectively. * indicates conserved amino acids upon ClustalW alignment.